【作者】 范寰；

【导师】 元英进；

【作者基本信息】 天津大学 ， 生物化工， 2007， 博士

【摘要】 为了研究稀土元素促进长春花悬浮培养细胞次生代谢产物的作用机理,以Ce⁴⁺作为诱导子,建立了1mMCe⁴⁺诱导长春花细胞悬浮培养体系,研究了1mMCe⁴⁺诱导作用下长春花细胞的活性氧、MAPK途径、PAL、次生代谢产物以及胞内金属离子的变化,试图从信号转导角度探索Ce⁴⁺诱导植物细胞次生代谢产物合成的基本规律。在研究Ce⁴⁺诱导作用下长春花细胞体系中H₂O₂的变化及其作用中发现Ce⁴⁺诱导长春花细胞出现了H₂O₂迸发现象;H₂O₂清除剂CAT的加入降低了Ce⁴⁺诱导的长春花细胞PAL的活化和长春质碱的合成;研究表明Ce⁴⁺诱导H₂O₂的产生先于长春质碱合成的激活,由此看出Ce⁴⁺诱导H₂O₂的产生处于Ce⁴⁺诱导长春质碱合成的上游。外加H₂O₂也能诱导细胞PAL的激活和长春质碱的合成,证明H₂O₂是Ce⁴⁺诱导长春花细胞次生代谢所必需的。研究还发现只是低浓度的Ce⁴⁺（0.5mM¹mM）促进长春花细胞H₂O₂的产生和长春质碱的合成。应用JNK、ERK和p38等途径特异性抑制剂的实验表明: JNK、ERK和p38等MAPK途径参与了Ce⁴⁺诱导长春花细胞体系次生代谢产物合成的过程,而只有ERK和p38途径参与了Ce⁴⁺诱导长春花细胞H₂O₂迸发的过程,但在H₂O₂迸发的下游JNK、ERK和p38等多条MAPK途径参与了将H₂O₂信号分子的传递调控次生代谢产物的合成。应用光谱学方法研究了不同浓度Ce⁴⁺对离体的过氧化氢酶（CAT）活力和构象的影响,发现0.01mM Ce⁴⁺在提高CAT活力的同时使其α-螺旋含量增加,β-折叠含量减小,而0.05 mM～0.2 mM Ce⁴⁺对CAT活性和二级结构的作用则相反。运用电感耦合等离子发射光谱仪对1mMCe⁴⁺诱导作用下的长春花细胞胞内的Ca²⁺、Mg²⁺、Zn²⁺等金属离子进行了测定,分析了在不同的诱导时间金属离子含量的变化和吲哚生物总碱合成之间的关系。研究发现1mMCe⁴⁺促进长春花细胞次生代谢产物合成的同时引起了细胞外Ca²⁺的内流和细胞内K +的外渗。更多还原

【Abstract】 The effect mechanism of rare earth on secondary metabolism productions in suspension cultured Catharanthus roseus was investigated by addition of 1mM Ce⁴⁺ to cultures. The changes of the activated oxygen species, mitogen-activated protein kinase （MAPK）, PAL and secondary metabolites were studied in detail. We attempted to explain the mechanism of secondary metabolites production in Ce⁴⁺ induced Catharanthus roseus.The changes and physiological role of H₂O₂ in Catharanthus roseus induced by Ce⁴⁺ were investigated. H₂O₂ burst after addition of Ce⁴⁺. PAL activation and the catharanthine production decreased significantly while CAT, a H₂O₂ scavenger, was added into cultures. Moreover, H₂O₂ generation was earlier than the catharanthine synthesis. We can conclude that H₂O₂ was the up-stream signal in catharanthine synthesis regulation. The production of catharanthine also increased after addition of H₂O₂. These results indicated that H₂O₂ play a key role in PAL activation and catharanthine synthesis. We also found that the concentration of Ce⁴⁺ strongly influenced H₂O₂ concentration and catharanthine production. Meanwhile, there was a saturated effect according to Ce⁴⁺ concentration.Signal transduction pathway that Ce⁴⁺ induced secondary metabolites synthesis in Catharanthus roseus was investigated by the addition of harmacological inhibitors. JNK、ERK and p38 were all activated in secondary metabolites synthesis induced by Ce⁴⁺. However, only ERK and p38 were activated in H₂O₂ generation induced by Ce⁴⁺. H₂O₂ signal was transduced by JNK、ERK and p38 protein kinase pathway to activated the secondary metabolites synthesis.The effects of Ce⁴⁺ on CAT activation and conformation were investigated by uv-visible spectra, fluorescent emission spectra, synchronous fluorescence, ultraviolet differential spectra, circular dichroism, FT-IR. The CAT conformation was related to the Ce⁴⁺ concentration.α-helix andβ-fold in CAT increase and decrease at lower concentration of Ce⁴⁺, respectively. However, the results reversed at higher concentration of Ce⁴⁺. These changes were related with Ce⁴⁺ concentration.Intracellular contents of Ca²⁺, Mg²⁺, Zn²⁺, Mn²⁺, Cu²⁺, Fe²⁺ and K+ were analyzed by an inductively coupled plasma-optical emission spectrometry and the relationships between ions contents and total alkaloid in cells at different induced time were investigated. The results showed that the secondary metabolism productions synthesis induced by 1mM Ce⁴⁺ was companying the Ca²⁺ influx and K+ efflux.更多还原